Modulation of antigen immunogenicity by deleting epitopes recognized by nkt cells

ABSTRACT

The invention describes a method and compounds for the prevention of immune responses towards allofactors, towards viral vectors used for gene therapy and gene vaccination, towards proteins to which subjects are naturally exposed, towards genetically-modified organisms and towards undesirable effects related to vaccine administration for allergic or infectious diseases.

FIELD OF THE INVENTION

The present invention relates to peptides or polypeptides to decreasenatural immunogenicity or induce tolerance and their use in preventingimmune responses elicited towards allofactors, towards viral vectorsused for gene therapy or gene vaccination, towards environmentalantigens used in food or feed or to which subjects are exposed byinhalation or contact, or to decrease side effects associated withvaccination by allergens or infectious agents.

BACKGROUND OF THE INVENTION

In many instances, administration of proteins for therapeutic purposesresults in immune response against the therapeutic agents, whichprecludes any further administration of said therapeutic agent. Examplesof this are provided by administration of factor VIII of the coagulationpathway in subjects affected by hemophilia A: about a third of treatedsubjects produce anti-factor VIII antibodies inhibiting the function offactor VIII. Administration of enzymes such as alpha-galactosidase insubjects suffering from deficiency in glycogen metabolism is likewisefollowed by an immune response against the therapeutic agent precludingany further administration. A third example is provided by antibodiesused as therapeutic agents and directed towards lymphocyte surfacemolecule, cytokines or cytokine receptors. Overall, it is highlydesirable to find new therapeutic approaches which would preventimmunization against therapeutic agents.

The use of viral vectors for gene therapy or gene vaccination isseverely restricted by the fact that such viral vectors elicit an immuneresponse which results in rapid elimination of cells transduced by thevector and rapid loss of transgene expression. Viral vectors elicitactivation of the innate and adaptive immune response with variabledegree of intensity depending of the nature of the viral vector. Thus,adenovirus vectors strongly stimulate innate immune responses followedby an adaptive immune response. Adeno-associated viral vectors trigger aweaker innate response but elicit antibody production. The potential ofboth gene therapy and gene vaccination is huge. A therapeutic approachby which the innate and/or the adaptive response towards viral vectorswould represent a highly significant progress in the field.

Many subjects suffer from response elicited against proteins to whichthey are naturally exposed. Allergens, either airborne or from food,elicit reactions such as allergic rhinitis and asthma, urticaria, eczemaand anaphylactic reaction. The reason as to why said subjects presentsuch reactions is only partially elucidated. It would be of much benefitto reduce the natural immunogenicity of proteins a diverse as foodallergens, wheat causing celiac disease or products such as enzymes towhich subjects are exposed for professional reasons.

In vaccination strategies for either allergic diseases or againstinfectious agents, the therapeutic efficacy is often limited by sideeffects which are elicited by the proinflammatory properties ofallergens or infectious agents. Such inflammatory effects preclude theuse of higher doses of vaccines, and therefore of vaccine efficacy, andorientate the immune response towards unwanted cellular response, suchas delayed-type reaction, and production of antibodies of an isotypewhich is not optimal for the condition considered. A better control ofinflammation in vaccination would offer a much more efficient modulationof the immune response whilst reducing side effects related toinflammation.

Natural killer T (NKT) cells constitute a distinct lineage ofnon-conventional T lymphocytes that recognize antigens presented by thenon-classical MHC complex molecule CD1d. Two subsets of NKT cells arepresently described. Type 1 NKT cells, also called invariant NKT cells(iNKT), are the most abundant. They are characterized by the presence ofan alpha-beta T cell receptor (TCR) made of an invariant alpha chain,Valpha14 in the mouse and Valpha24 in humans. This alpha chain isassociated to a variable though limited number of beta chains. Type 2NKT cells have an alpha-beta TCR but with a polymorphic alpha chain.However, it is apparent that other subsets of NKT cells exist, thephenotype of which is still incompletely defined, but which share thecharacteristics of being activated by glycolipids presented in thecontext of the CD1d molecule.

NKT cells typically express a combination of natural killer (NK) cellreceptor, including NKG2D and NK1.1. NKT cells are part of the innateimmune system, which can be distinguished from the adaptive immunesystem by the fact that they do not require expansion before acquiringfull effector capacity. Activation of NKT cells results in variouseffects. Such cells release preformed mediators, including a large arrayof cytokines (including interleukin (IL)-4, interferon (IFN)-gamma,IL-21 and IFN-alpha) which provide help to B cells for the production ofantibodies and it has been suggested that the release of cytokines couldalso influence CD4+ T cells (Burrows et al Nature Immunology 2009, 10:669-671). In the context of the present invention, the prevention ofboth B cell activation and of major histocompatibility (MHC) classII-restricted CD4+ T cells activation is deemed to play a role inreducing or abolishing protein immunogenicity.

The recognition unit for NKT cells, the CD1d molecule, has a structureclosely resembling that of the MHC class I molecule, including thepresence of beta-2 microglobulin. It is characterized by a deep cleftbordered by two alpha chains and containing highly hydrophobic residues,which accepts lipid chains. The cleft is open at both extremities,allowing to accommodate longer chains. The canonical ligand for CD1d isthe synthetic alpha galactosylceramide (alpha GalCer). However, manynatural alternative ligands have been described, including glyco- andphospholipids, the natural lipid sulfatide found in myelin, microbialphosphoinositol mannoside and alpha-glucuronosylceramide. The presentconsensus (see reviews, such as Matsuda et al, Current Opinion inImmunology 2008, 20:358-368 and Godfrey et al, Nature reviews Immunology2010, 11: 197-206) is that CD1d binds only ligands containing lipidchains, or in general a common structure made of a lipid tail which isburied into CD1d and a sugar residue head group that protrudes out ofCD1d.

Peptides are not deemed to be able to activate NKT cells throughpresentation by CD1d. It was, however, suggested that long hydrophobicpeptides containing bulky aminoacid residues could bind to CD1d (Castanoet al, Science 1995, 269: 223-226). Observations carried out using phagedisplay libraries expressing random sequence peptides with no definedphysiological relevance, allowed establishing a theoretical consensusmotif (Castano et al, Science 1995, 269: 223-226 and see below).

In fact, Castano et al show that the cells which are activated are CD8+T cells, namely MHC class I restricted cells, and not NKT cells. Thesefindings teach the one skilled in the art that there is no evidence thathydrophobic peptides are presented by CD1d molecules. The physiologicalrelevance of the claims made by Castano et al was further questioned dueto the inability to elicit NKT cells under conventional immunizationprotocols (Matsuda et al, Current Opinion in Immunology 2008, 20:358-368and Brutkiewicz Journal of Immunology 2006, 177: 769-775). Artificialsystems such as immunization with cells transfected to overexpress CD1dand loaded in vitro with an ovalbumin-derived peptide were able toelicit NKT cells. Likewise, intradermal immunization with plasmid DNAtogether with murine CD1d and costimulatory molecules induce cytolyticCD1d-restricted T cells (Lee et al, Journal of Experimental Medicine1998, 187: 433-438). Hydrophobic peptides containing a structural motifmade of an aromatic residue in position P1 and P7, and an aliphaticchain in position P4 were claimed by Castano et al (Science 269: 223,1995) to contain a core motif for CD1d binding epitopes. As describedabove, the conclusions reached by Castano et al are not supported bydata.

We made the unexpected finding that peptides encompassing a hydrophobicaminoacid sequence are in fact capable of eliciting activation of NKTcells.

If epitopes from proteins administrated for therapeutic purposes, or towhich subjects are normally exposed, or when gene therapy or genevaccination is carried out, or administered in the context ofvaccination for allergic or infectious diseases bind to CD1d and therebyactivate NKT cells, then alteration of said proteins by mutations and/ordeletions to eliminate said epitopes would be highly desirable toprevent immunogenicity.

Identification of such epitopes followed by mutation, addition ordeletion of aminoacids to prevent activation of NKT cells forms thebasis of the present invention.

SUMMARY OF THE INVENTION

The present invention relates to the use of peptides or polypeptides forthe treatment of immune responses elicited towards allofactors in asubject by preventing such immune response towards said allofactors.

The present invention also relates to the use of peptides orpolypeptides for the treatment of immune responses elicited towardsviral vectors used for gene therapy or gene vaccination in a subject bypreventing such immune response towards said viral vectors.

The present invention also relates to the use of peptides orpolypeptides made by genetically-modified organisms for the preventionin a subject of immune responses elicited by exposure to naturalproteins.

The present invention further relates to the use of peptides orpolypeptides for vaccination purposes when activation of innate immunityis detrimental.

The present invention also relates to methods to identify proteins whichcarry CD1d binding epitopes and to eliminate such epitopes by aminoacidsubstitution or deletion.

We made the unexpected finding that a significant proportion of peptidesor polypeptides carried aminoacid sequences which allow them to bind andto be presented by CD1d determinants for activation of natural killer T(NKT) cells. Activation of such cells results in release of cytokinesand, in some cases, in acquisition or increase of cytolytic properties.

The present invention relates in one aspect to the use of at least oneisolated peptide or polypeptide used as an allofactor, which has beenmodified to eliminate at least one hydrophobic amino acid residueinvolved in the formation of an epitope recognized by NKT cells, as amedicament for preventing in a subject immune responses to saidallofactor.

The present invention also relates in one aspect to the use of at leastone isolated peptide or polypeptide used as a viral vector for genetherapy or gene vaccination, which has been modified to eliminate atleast one hydrophobic amino acid residue involved in the formation of anepitope recognized by NKT cells, as a medicament for preventing in asubject immune responses to said viral vectors.

The present invention also relates in one aspect to the use of at leastone isolated peptide or polypeptide produced by a genetically-modifiedorganism, said peptide or polypeptide being modified to eliminate atleast one hydrophobic amino acid residue involved in the formation of anepitope recognized by NKT cells, as a medicament for preventing in asubject immune responses to natural exposure to said peptides orpolypeptides.

The present invention further relates in one aspect to the use of atleast one isolated peptide or polypeptide used as a vaccine, which hasbeen modified to eliminate at least one hydrophobic amino acid residueinvolved in the formation of an epitope recognized by NKT cells, as amedicament for preventing in a subject an unwanted or inappropriateimmune response to said vaccine.

In a further aspect, the invention also covers the use of at least oneisolated peptide or polypeptide used as an allofactor, a viral vector, agenetically-modified organism or a vaccine, which has been modified toeliminate at least one hydrophobic amino acid residue involved in theformation of an epitope recognized by NKT cells, as a medicament forpreventing in a subject activation, cytokine production, cytolyticactivity and suppressive activity on adaptive immune responses carriedby CD4+ NKT cells in said subject.

The present invention relates to hydrophobic peptides or polypeptidesencompassing at least one CD1d-restricted T cell epitope, in whichaminoacids positioned as anchoring residues to CD1d are replaced byalternative aminoacids, or deleted, which results in a loss orsignificant reduction of binding to CD and thereby of NKT cellactivation.

The structure of the CD1d molecule indicates that hydrophobic aminoacidresidues are required to occupy the two hydrophobic pockets located atthe extremities of the CD1d cleft and that an aliphatic residue shouldoccupy the position in the middle of the cleft. Therefore, as a generalexample of CD1d binding sequence, the motif [FW]-xx-[ILM]-xx-[FWTH] canbe used in which [FW] indicates that either F or W can occupy the firstanchoring residue (P1), that the P4 position can be occupied by eitherI, L or M and that P7 can be occupied by F, W, T or H. x in this generalmodel motif stands for any aminoacid. It should be clear for the oneskilled in the art that various combinations of these aminoacid residuesare possible. In a particular embodiment the general model motif can bepresented as a reverted sequence such as [FWTH]-xx-[ILM]-xx-[FW]. In yetanother particular embodiment at least one aminoacid is added within theCD1d binding motif, which disrupts the motif, prevents its capacity tobind to CD1d and thereby its capacity to activate NKT cells.

The present invention further relates more particularly to peptides orpolypeptides wherein F, W, T, H or Y in positions P1 and P7 are replacedby a non-natural amino acid (for example a D-aminoacid) or by an organiccompound.

In any of the above uses said allofactor may be any peptide orpolypeptide used: (1) for replacement therapy for coagulation defects orfibrinolytic defects, including factor VIII, factor IX andstaphylokinase; (2) hormones such as growth hormone or insulin; (3)cytokines and growth factors, such as interferon-alpha,interferon-gamma, GM-CSF and G-CSF; (4) antibodies for the modulation ofimmune responses, including anti-IgE antibodies in allergic diseases,anti-CD3 and anti-CD4 antibodies in graft rejection and a variety ofautoimmune diseases, anti-CD20 antibodies in non-Hodgkin lymphomas; (5)erythropoietin in renal insufficiency and; (6) genetically modifiedantigens.

In any of the above uses said viral vector may be any peptide orpolypeptide of RNA viruses (gamma-retroviruses and lentiviruses) or DNAviruses (adenoviruses, adeno-associated viruses, herpes viruses andpoxviruses).

In any of the above uses said genetically-modified organism may be anyorganism of plant or animal origin, which is used as food or feed, forproducing crops or manufacture material, or for producing transgenicanimals for food or feed, or stock breeding.

In any of the above, said peptide or polypeptide used for vaccinationmay be from allergens or from infectious agents, including viruses,bacteria and parasites. Allergens may be airborne allergens such asthose derived from house dust mite, from pollens or from domesticanimals, food allergens such as peanut, ovalbumin, cereals, fruits andlegumes, and contact antigens such as latex. Diseases characterizingallergen sensitization include allergic asthma, allergicrhino-sinusitis, anaphylactic shock, urticaria, atopic dermatitis andcontact dermatitis.

The present invention also relates to methods for identifying peptidesor polypeptides activating NKT cells and eliminates such activation byaltering CD1d binding epitopes by substitution, addition or deletion ofaminoacids. Said methods comprise the steps of incubating said peptideor polypeptide with cells carrying CD1d, followed by addition of apopulation of polyclonal NKT cells and determination of activation ofsaid NKT cells.

The invention further encompasses isolated viral vectors characterizedin that they comprise at least one peptide or polypeptide of anallofactor modified by substitution or deletion of at least onehydrophobic aminoacid, or at least one peptide or polypeptide from anallergen or from a infectious agent modified by substitution or deletionof at least one hydrophobic aminoacid residue. It should be understoodthat the viral vector itself may also be modified by substitution ordeletion of hydrophobic aminoacid residues.

DEFINITIONS

The term “peptide” when used herein refers to a molecule comprising anamino acid sequence of between 2 and 200 amino acids, connected bypeptide bonds, but which can in a particular embodiment comprisenon-amino acid structures (like for example a linking organic compound).Peptides according to the invention can contain any of the conventional20 amino acids or modified versions thereof, or can containnon-naturally occurring amino acids incorporated by chemical peptidesynthesis or by chemical or enzymatic modification. The term“polypeptide” when used herein refers to generally longer peptides orproteins.

The term “epitope” when used herein refers to one or several portions(which may define a conformational epitope) of a protein which is/arespecifically recognized and bound by an antibody or a portion thereof(Fab′, Fab2′, etc.) or a receptor presented at the cell surface of a Bor T cell lymphocyte, and which is able, by said binding, to induce animmune response.

The term “antigen” when used herein refers to a structure of amacromolecule comprising one or more hapten(s) and/or comprising one ormore T cell epitopes. Typically, said macromolecule is a protein orpeptide (with or without polysaccharides) or made of proteic compositionand comprises one or more epitopes; said macromolecule can hereinalternatively be referred to as “antigenic protein” or “antigenicpeptide”.

The term “allergen” refers to a specific subset of antigen characterizedby its capacity to elicit antibodies of the IgE isotype in predisposedindividuals.

The term “T cell epitope” or “T-cell epitope” in the context of thepresent invention refers to a dominant, sub-dominant or minor T cellepitope, i.e., a part of an antigenic protein that is specificallyrecognized and bound by a receptor at the cell surface of a Tlymphocyte. Whether an epitope is dominant, sub-dominant or minordepends on the immune reaction elicited against the epitope. Dominancedepends on the frequency at which such epitopes are recognized by Tcells and able to activate them, among all the possible T cell epitopesof a protein. In particular, a T cell epitope is an epitope bound by MHCclass I or MHC class II molecules.

The term “NKT cell epitope” refers to a part of an antigenic proteinthat is specifically recognized and bound by a receptor at the cellsurface of a T lymphocyte. In particular, a NKT cell epitope is anepitope bound by CD1d molecules.

The term “CD4+ effector cells” refers to cells belonging to theCD4-positive subset of T-cells whose function is to provide help toother cells, such as, for example B-cells. These effector cells areconventionally reported as Th cells (for T helper cells), with differentsubsets such as Th0, Th1, Th2, and Th17 cells.

The term “NKT cells” refers to cells of the innate immune systemcharacterized by the fact that they carry receptors such as NK1.1 andNKG2D, and recognize epitopes presented by the CD1d molecule. In thecontext of the present invention, NKT cells can belong to either thetype 1 (invariant) or the type 2 subset.

The “CD1d molecule” refers to a non-MHC derived molecule made of 3 alphachains and an anti-parallel set of beta chains arranged into a deephydrophobic groove opened on both sides and capable of presentinglipids, glycolipids or hydrophobic peptides to NKT cells.

The term “immune disorders” or “immune diseases” refers to diseaseswherein a reaction of the immune system is responsible for or sustains amalfunction or non-physiological situation in an organism. Immunedisorders in the context of the present invention refer to pathologyinduced by infectious agents and tumor surveillance.

The term “allofactor” refers to a protein, peptide or factor (i.e. anymolecule) displaying polymorphism when compared between two individualsof the same species, and, more in general, any protein, peptide orfactor that induces an (alloreactive) immune response in the subjectreceiving the allofactor. By extension, allofactors also includegenetically-modified proteins used for feeding.

The term “alloantigen” or “allograft antigen” when used herein refer toan antigen derived from (shed from and/or present in) a cell or tissuewhich, when transferred from a donor to a recipient, can be recognizedand bound by an antibody of B or T-cell receptor of the recipient.Alloantigens are typically products of polymorphic genes. An alloantigenis a protein or peptide which, when compared between donor and recipient(belonging to the same species), displays slight structural differences.The presence of such a donor antigen in the body of a recipient canelicit an immune response in the recipient. Such alloreactive immuneresponse is specific for the alloantigen.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides ways to prevent, in a subject, an immuneresponse towards allofactors, towards viral vectors used for genetherapy or gene vaccination, towards proteins used for food or feed,towards proteins to which said subject is exposed by inhalation or bystings, or to prevent, in a subject, an undesirable activation of innateimmunity in the use of vaccines towards allergens or infectious agents.

In particular, the invention provides ways to prevent the expansion andfunctional activity of CD4+ NKT cells. Such cells are usually classifiedinto two distinct subsets, namely type 1 for NKT cells carrying aninvariant TCR alpha chain (Valpha14 in the mouse, Valpha24 in humans),or type 2 NKT cells which present with a diverse alpha chain repertoire.However, recent evidence has suggested that alternative subsets of NKTcells which do not fit in the type 1 or type 2 category. It is thepurpose of the present invention to include these non conventional NKTcells, provided they carry the CD4 co-receptor. Upon presentation of anantigen bound to CD1d, NKT cells are rapidly activated and secrete anumber of cytokines thought to be determinant to influence other cellsfrom both the innate and adaptive immune systems. In some circumstances,said activated NKT cells acquire or increase cytotoxic properties. Inyet additional circumstances, said activated NKT cells suppress orreduce the elicitation of an adaptive immune response by interactionwith class II-restricted CD4+ T cells.

In the context of the present invention, we made the unexpectedobservation that peptides can be presented by the CD1d molecule. Acharacteristic of the CD1d molecule is that it is made of twoanti-parallel alpha chains forming a cleft sitting atop of a platformmade of two anti-parallel beta chains. The cleft is narrow and deep andaccept only hydrophobic residues, classically deemed to be only lipids.The cleft can accommodate a sequence of 7 aminoacids characterized as ahydrophobic residue in position (P)1 and 7, and an aliphatic residue inP4.

P1 is an obligate hydrophobic residue, such as F, W, H or Y. However, P7is permissive and can contain alternative residues provided they are notpolar. Residues in P4 are preferably aliphatic but are optional. Ageneral sequence for a CD1d binding motif is therefore[FWTHY]-X₂X₃-[ILMV]-X₅X₆-[FWTHY]. It should however be clear for thoseskilled in the art that the motif is symetrical and that P7 can beconsidered as P1, and P1 can be considered as P7. The general sequenceof a CD1d binding motif is provided here as a general indication withoutany limiting intention. Peptides and polypeptides considered forapplication of the present invention are defined according to theircapacity to activate NKT cells by presentation into CD1d molecule.

Hydrophobic peptides or polypeptides capable of activating NKT cellsand, consequently, carrying a CD1d-binding motif are found inallofactors, viral vectors, proteins used for food or feed, proteins towhich said subject is exposed by inhalation or by stings,genetically-modified proteins and allergens, thereby endowing saidallofactor, viral vector, genetically-modified protein or allergen withthe capacity to activate CD4+ NKT cells.

The present invention relates to the production of peptides orpolypeptides containing CD1d binding motif(s), which confer them withthe capacity to activate NKT cells and which are modified bysubstitution of hydrophobic residues in P1 and/or P7, with, optionally,substitution or deletion of aliphatic residues in P4, or any combinationof these, which results in a loss or significant reduction of thecapacity of peptides or polypeptides to bind to CD1d and thereby resultsin a loss or significant reduction of said peptides or polypeptides toactivate NKT cells.

In a more particular embodiment, F, W, T, H or Y in positions P1 and/orP7 are replaced by a non-hydrophobic aminoacid residue, or, optionally,I, L, M or V in position P4 is replaced by a non-aliphatic residue, orany combination of these.

In yet another particular embodiment, hydrophobic residues located inposition P1 and/or P7, or, optionally, aliphatic residues located in P4,or any combination of these, are replaced by at least one non-naturalaminoacid different from non-natural F, W, T, H, Y, or by a non-aromaticorganic compound.

In yet another particular embodiment at least one aminoacid is addedwithin the CD1d binding motif, in any location within the P1 to P7sequence, which disrupts the motif, prevents its capacity to bind toCD1d and thereby its capacity to activate NKT cells.

In a preferred embodiment, non-natural aminoacids are D-aminoacids.

The present invention also relates to the production of peptides orpolypeptides containing CD1d binding motif(s), which confer them withthe capacity of activate NKT cells, and which are modified by deletionof hydrophobic residues in P1 and/or P7, or, optionally, by deletion ofaliphatic residues in P4, or any combination of these, which results ina loss or significant reduction of the capacity of peptides orpolypeptides to bind to CD1d and thereby results in a loss orsignificant reduction of said peptides or poylpeptides to activate NKTcells.

Upon administration to a subject, such peptides or polypeptides are notloaded on CD1d and thereby are prevented from activating NKT cells.

In a further aspect, the invention also covers the use of at least oneisolated peptide or polypeptide comprising at least one substitution ordeletion of F, W, T, H or Y in positions P1 or P7 for preventing in asubject an immune response towards allofactor administration, viralvector administration, proteins to which said subject is exposed byfood, feed, systemic or inhalation route, or allergens or infectiousagents used for vaccination purposes.

In yet a further aspect, the invention covers the use of at least oneisolated peptide or polypeptide comprising at least one substitution ordeletion of F, W, T, H or Y in positions P1 or P7 for preventing in asubject the activation of NKT cells towards allofactor administration,viral vector administration, proteins to which said subject is exposedby food, feed, systemic or inhalation route, or some allergens orinfectious agents used for vaccination purposes.

In yet a further aspect, the invention also covers the use of at leastone isolated peptide or polypeptide comprising at least one substitutionor deletion of F, W, T, H or Y in positions P1 or P7 as a medicament forpreventing in a subject an immune response towards allofactoradministration, viral vector administration, proteins to which saidsubject is exposed by food, feed, systemic or inhalation route,genetically modified peptides or polypeptides or some allergens orinfectious agents used for vaccination purposes.

The number of CD1 d binding motifs when present in a peptide orpolypeptide, is very limited. Examples of such peptides or polypeptidesare provided below. Typically a polypeptide presents one to five ofthese motifs.

An additional advantage of the present invention is that the CD1dmolecule presents a very limited degree of polymorphism. It is thereforeobvious for the one skilled in the art that the same aminoacidsubstitutions, addition or deletions according to the present inventionprovide peptides or polypeptides useful for all or a large majority ofsubjects. This is in sharp contrast with peptide or polypeptide motifsbinding to MHC class II molecules, wherein a large number of peptidescan be delineated which contain the appropriate sequence. This is due tothe minimum constraints imposed to MHC class II binding peptides and tothe large polymorphism of class II molecules.

Peptides and polypeptides which are the object of the present inventionare identified as follows:

(1) a peptide or polypeptide aminoacid sequence is, optionally,evaluated for the presence of at least one CD1d motif containing anhydrophobic residue in P1 and P7, and an aliphatic residue in P4. Ageneral sequence such as [FWTHY]-X₂X₃-[ILMV]-X₅X₆-[FWTHY] can be usedfor using algorithms well known in the art such as

http://expasy.org/tools/scanprosite/

This general sequence should be considered as a tool to help identifyingwhich sequence(s) in said peptide or polypeptide contain a motif whichcould enable said peptide or polypeptide to activate NKT cells.

(2) the capacity of the peptide or polypeptide to bind to CD1d and toactivate NKT cells is tested in vitro using a cell line expressing theCD1d molecule. Examples of such cell lines are known in the art (forinstance JAW S2 cells). In a preferred embodiment, the cell line is notpresenting MHC class II molecules and is transduced for hyperexpressionof CD1d using a viral vector containing the DNA sequence of CD or anyother means known in the art to introduce a gene in a cell. Methods forcell transduction are known in the art. The cell line is loaded inculture with the peptide or polypeptide, or with a synthetic peptideencompassing the corresponding sequence. Such synthetic peptides areeasily produced by synthesis, using for instance the fmoc solid phasesynthesis well known in the art. Efficient presentation of the peptide,polypeptide or corresponding synthetic peptide by the CD1d molecule isthen evaluated by measuring the activation of NKT cells. Such cells canbe obtained from peripheral blood by, for instance, magnetic sorting andmaintained in culture with stimulants such as alpha-gal-ceramide, in thepresence of cytokines such as IL-2, IL-15 or IL-7. These methods aredescribed in the art (see for instance Godfrey et al, Nature Reviews.Immunology 2010, 11: 197-206). Activation of NKT cells is assessed usingmethods such as evaluation of cytokine production.

Alternatively, peptides actually presented by APC in CD1d molecules canbe eluted and separated by various chromatography methods. Fulldescription of such methodology will be found in Scott et al, Immunity,12: 711-720, 2000. Said peptides are then sequenced to identify whichaminoacid residues are located in P1 and P7.

Alternatively, said synthetic peptides can be loaded on tetramers of theCD1d molecule to detect NKT cells specific for such peptide. Onepossibility is to use fluorescence-labeled tetramers and detection usinga fluorescence-activated cell sorting system (facs).

(3) the aminoacid sequences identified as being able to activate NKTcells and, optionally, identified by algorithms, are then modified byeither substitution or deletion. In a preferred embodiment, F, W, T, Hor Y in positions P1 and/or P7 are replaced by at least one aminoaciddifferent from F, W, T, H, Y. Natural aminoacids can be modified bypost-transcriptional modifications or substituted with chemical groupssuch as methyl groups. In another preferred embodiment, F, W, T, H or Yin positions P1 and/or P7 are replaced by any suitable alternativenon-natural aminoacid. Examples of non-natural aminoacid residues areD-aminoacids. In yet another embodiment, F, W, T, H or Y in positions P1and/or P7 are replaced by at least one aminoacid different from F, W, T,H, Y. In another preferred embodiment, F, W, T, H or Y in position P1 isreplaced by at least one aminoacid different from F, W, T, H, Y, by anysuitable alternative non-natural aminoacid or by a non-aromatic organiccompound. Such aminoacid substitution is obtained using methods wellknown in the art. In yet a further preferred embodiment, F, W, T, H or Yin position P1 is deleted. In yet another embodiment, F, W, T, H or Y inpositions P1 and P7 are deleted. Methods to carry out said deletions arewell known in the art. In yet another particular embodiment at least oneaminoacid is added within the CD1d binding motif, in any location withinthe P1 to P7 sequence.

According to the present invention medicaments are envisaged for thetreatment of diseases wherein administration of allofactors arerequired, such as in:

(1) congenital or acquired deficiency in factors associated withcoagulation (such as factor VIII, factor IX or factor X) orfibrinolysis, with defect in enzymes associated with the metabolism ofpolysaccharides or glycogen (such as in Pompe disease), or with defectin hormone production (such as insulin in diabetes or growth hormone innanism)

(2) acute or chronic situations wherein it is advantageous to administera curative agent, such as thrombolytic agents including staphylokinaseand microplasmin,

(3) disorders of the immune system in which it is required to administercytokines (or their receptor) or growth factors (such asinterferon-alpha, interferon-beta, interferon-gamma, G-CSF, GM-GSF, KGFor erythropoietin)

(4) diseases characterized by chronic inflammation or inappropriateimmune responses, wherein therapeutic antibodies should be administered,including anti-tumor necrosing factor, anti-CD3 or anti-CD4 antibodiesin autoimmune diseases and graft rejection, antibodies to lymphocytesurface markers (such as anti-CD20 antibodies in non-Hodgkin lymphomas),or antibodies to factor VIII in the prevention of thrombosis. The listof therapeutic antibodies is growing fast and the present inventionintends to cover the use of any antibodies used for therapeutic purposesin general.

According to the present invention medicaments are also envisaged foruse in gene therapy and gene vaccination, wherein viral vectors areutilized and wherein the immune response against said vectors precludestransgene expression.

According to the present invention medicaments are also envisaged fordiseases elicited by exposure to environmental proteins, such as:

(1) proteins to which said subject is exposed by food or feed. Examplesof these are cereals such as wheat, maize, rice, soybean and colza,vegetables such as potato and beetroot, fruits such as rosacea, nuts,and avocado, enzymes, anti-viral or anti-bacterial drugs.

(2) proteins towards which the subject is exposed by inhalation,systemic route or by stinging. Examples of these are allergic reactionsto pollens, contact reaction to latex or hymenoptera stings.

According to the present invention medicaments are also envisaged forimmunization (vaccination) such as:

(1) vaccination against allergens

(2) vaccination against infectious agents, including viruses, bacteriaand parasites

In both these circumstances it may be advantageous to prevent anactivation of the innate immune system so as to prevent excess ofinflammation and its detrimental consequences on the result of saidvaccination. Another advantage in the setting of vaccination toallergens or infectious agents is that the elimination of NKT cellactivation prevents the suppressive effect of activated NKT cells on thedevelopemnt of an adaptive response against said allergens or saidinfectious agents.

It should be recognized that the above list is not exhaustive and thatthe invention intends to cover newly-introduced products such asantibodies, cytokines, growth factors or peptides and polypeptides usedfor replacement in congenital or acquired deficiencies, andgenetically-modified proteins.

It should be understood that any of the peptides or polypeptides listedabove may be administered in the form of gene for transgenesis, whichmay be carried out using viral vectors or other means known by thoseskilled in the art. In such a case, the viral vector itself may bemodified according to the present invention by eliminating CD1d bindingmotifs.

The medicament of the invention is usually, though not necessarily, a(pharmaceutical) formulation comprising as active ingredient at leastone of the peptides or polypeptides of the invention or a genetherapeutic vector capable of expressing said peptides or polypeptides.Apart from the active ingredient(s), such formulation will comprise atleast one of a (pharmaceutically acceptable) diluent.

A notable exception to this rule is the use of proteins fromgenetically-modified organisms for food or feed, exposure by inhalationor by the systemic route.

In general, administration of peptides or polypeptides of the inventionprevents activation of the innate immune system, more particularlyactivation of NKT cells, more particularly the production of cytokinesassociated with NKT cell activation.

The route of administration for peptides or polypeptides of the presentinvention may vary according to the indication and/or the nature of thepeptides or polypeptides. Examples are intravenous injection ofcoagulation factors, subcutaneous injection of insulin and oraladministration of genetically-modified proteins. The present inventionintends to cover all other possible routes of administration such asintranasal, sublingual, percutaneous, intramuscular, intrarectal orintravaginal.

As explained in detail further on, the peptides or polypeptides of thepresent invention can be made by chemical synthesis, which furtherallows the incorporation of non-natural amino acids. The peptides orpolypeptides of the present invention can also be produced using methodsknow in the art for the production of recombinant proteins usingexpression systems such as bacterial cells, yeast cells, insect cells,plant cells or mammalian cells.

Another aspect of the present invention relates to methods forgenerating peptides and polypeptides of the present invention describedherein. Such methods include the identification of NKT-cell epitopesfrom allofactors, viral vectors, proteins to which said subject isexposed by food, feed, systemic or inhalation route, allergens orspecific infectious agents. Ways for in vitro and in silicoidentification NKT-cell epitopes are amply known in the art and someaspects are elaborated upon hereafter.

The identification of a NKT-cell epitope in the context of the presentinvention is known to a person skilled in the art. For instance, peptidesequences isolated from allofactors, viral vectors, proteins to whichsaid subject is exposed by food, feed, systemic or inhalation route,genetically-modified proteins, allergens or specific infectious agentsare tested by, for example, NKT cell biology techniques, to determinewhether the peptide sequences elicit a NKT cell response. Those peptidesequences found to elicit a NKT cell response are defined as having NKTcell stimulating activity. Mammal NKT cell stimulating activity canfurther be tested by culturing NKT cells obtained from an individualsensitized to an allofactor, viral vector, proteins to which saidsubject is exposed by food, feed, systemic or inhalation route,genetically-modified protein, allergen or specific infectious agent, anddetermining whether proliferation of NKT cells occurs in response to thepeptide/epitope as measured, e.g., by cellular uptake of tritiatedthymidine. Stimulation indices for responses by NKT cells topeptides/epitopes can be calculated as the maximum CPM in response to apeptide/epitope divided by the control CPM. A NKT cell stimulation index(S.I.) equal to or greater than two times the background level isconsidered “positive.” Positive results are used to calculate the meanstimulation index for each peptide/epitope for the group ofpeptides/epitopes tested. Non-natural NKT-cell epitopes can furtheroptionally be tested for their binding affinity to CD1d molecules. Thebinding of non-natural NKT-cell epitopes to CD1d molecules can beperformed in different ways. For instance, soluble CD1d molecules areobtained and made tetrameric by synthesis and/or chemical coupling. TheCD1d molecule is purified by affinity chromatography. Soluble CD1dmolecules are incubated with a biotin-labeled reference peptide producedaccording to its strong binding affinity for said CD1d molecule.Peptides to be assessed for CD1d binding are then incubated at differentconcentrations and their capacity to displace the reference peptide fromits CD1d binding site is calculated by addition of neutravidin. Methodscan be found in for instance in Texier et al., (2000) J. Immunology 164,3177-3184) for peptides presented by the MHC class II determinants, butthe method can easily be applied to CD1d-restricted NKT cell epitopes.The immunogenic peptides or polypeptides of the invention have a meanNKT cell stimulation index of greater than or equal to 2. An immunogenicpeptide having a NKT cell stimulation index of greater than or equal to2 is considered useful as a candidate to carry out the substitution ordeletion of hydrophobic aminoacid residues, or addition of aminoacidswithin the sequence of the CD1d binding motif, as described in thepresent invention.

If two or more aminoacid sequences which share an area of overlap in thenative peptide or polypeptide sequence are found to have human NKT cellstimulating activity, as determined by T cell biology techniques,mutation or deletion of hydrophobic aminoacid residues may be carriedout for residues belonging to one or to both of the sequences.

The peptides or polypeptides of the invention can be produced byrecombinant expression in, e.g., bacterial cells (e.g. Escherichiacoli), yeast cells (e.g., Pichia species, Hansenula species,Saccharomyces or Schizosaccharomyces species), insect cells (e.g. fromSpodoptera frugiperda or Trichoplusia ni), plant cells or mammaliancells (e.g., CHO, COS cells). The construction of the therefore requiredsuitable expression vectors (including further information such aspromoter and termination sequences) involves meanwhile standardrecombinant DNA techniques. Recombinantly produced peptides orpolypeptides of the invention can be derived from a larger precursorprotein, e.g., via enzymatic cleavage of enzyme cleavage sites insertedadjacent to the N- and/or C-terminus of the peptide or polypeptide,followed by suitable purification.

In view of the limited length of some of the peptides of the invention,they can be prepared by chemical peptide synthesis, wherein peptides areprepared by coupling the different amino acids to each other. Chemicalsynthesis is particularly suitable for the inclusion of e.g. D-aminoacids, amino acids with non-naturally occurring side chains or naturalamino acids with modified side chains such as methylated cysteine.Chemical peptide synthesis methods are well described and peptides canbe ordered from companies such as Applied Biosystems and othercompanies. Peptide synthesis can be performed as either solid phasepeptide synthesis (SPPS) or contrary to solution phase peptidesynthesis. The best-known SPPS methods are t-Boc and Fmoc solid phasechemistry which is amply known to the skilled person. In addition,peptides can be linked to each other to form longer peptides using aligation strategy (chemoselective coupling of two unprotected peptidefragments) as originally described by Kent (Schnolzer & Kent (1992) Int.J. Pept. Protein Res. 40, 180-193) and reviewed for example in Tam etal. (2001) Biopolymers 60, 194-205. This provides the potential toachieve protein synthesis which is beyond the scope of SPPS. Manyproteins with the size of 100-300 residues have been synthesizedsuccessfully by this method.

The physical and chemical properties of a peptide or polypeptide of theinvention (e.g. solubility, stability) is examined to determine whetherthe peptide is/would be suitable for use in therapeutic compositions.Typically this is optimized by adjusting the sequence of the peptide.Optionally, the peptide can be modified after synthesis (chemicalmodifications e.g. adding/deleting functional groups) using techniquesknown in the art.

The production of genetically-modified organisms relies on methods wellknown for those skilled in the art, including cloning, site-directedmutagenesis and growth.

The present invention also relates to nucleic acid sequences encodingthe peptides or polypeptides of the present invention and methods fortheir use, e.g., for recombinant expression or in gene therapy. Inparticular, said nucleic acid sequences are capable of expressingpeptides of the invention.

In gene therapy, recombinant nucleic acid molecules encoding thepeptides or polypeptides of the present invention can be used as nakedDNA or in liposomes or other lipid systems for delivery to target cells.Other methods for the direct transfer of plasmid DNA into cells are wellknown to those skilled in the art for use in human gene therapy andinvolve targeting the DNA to receptors on cells by complexing theplasmid DNA to proteins. In its simplest form, gene transfer can beperformed by simply injecting minute amounts of DNA into the nucleus ofa cell, through a process of microinjection. Once recombinant genes areintroduced into a cell, they can be recognized by the cell normalmechanisms for transcription and translation, and a gene product will beexpressed. Other methods have also been attempted for introducing DNAinto larger numbers of cells. These methods include: transfection,wherein DNA is precipitated with calcium phosphate and taken into cellsby pinocytosis; electroporation, wherein cells are exposed to largevoltage pulses to introduce holes into the membrane);lipofection/liposome fusion, wherein DNA is packed into lipophilicvesicles which fuse with a target cell; and particle bombardment usingDNA bound to small projectiles. Another method for introducing DNA intocells is to couple the DNA to chemically modified proteins. Adenovirusproteins are capable of destabilizing endosomes and enhancing the uptakeof DNA into cells. Mixing adenovirus to solutions containing DNAcomplexes, or the binding of DNA to polylysine covalently attached toadenovirus using protein crosslinking agents substantially improves theuptake and expression of the recombinant gene. Adeno-associated virusvectors may also be used for gene delivery into vascular cells. As usedherein, “gene transfer” means the process of introducing a foreignnucleic acid molecule into a cell, which is commonly performed to enablethe expression of a particular product encoded by the gene. The saidproduct may include a protein, polypeptide, anti-sense DNA or RNA, orenzymatically active RNA. Gene transfer can be performed in culturedcells or by direct administration into mammals. In another embodiment, avector comprising a nucleic acid molecule sequence encoding a peptideaccording to the invention is provided. In particular embodiments, thevector is generated such that the nucleic acid molecule sequence isexpressed only in a specific tissue. Methods of achievingtissue-specific gene expression are well known in the art, e.g., byplacing the sequence encoding an immunogenic peptide of the inventionunder control of a promoter, which directs expression of the peptidespecifically in one or more tissue(s) or organ(s). Expression vectorsderived from viruses such as retroviruses, vaccinia virus, adenovirus,adeno-associated virus, herpes viruses, RNA viruses or bovine papillomavirus, may be used for delivery of nucleotide sequences (e.g., cDNA)encoding peptides, homologues or derivatives thereof according to theinvention into the targeted tissues or cell population. Methods whichare well known to those skilled in the art can be used to constructrecombinant viral vectors containing such coding sequences.Alternatively, engineered cells containing a nucleic acid moleculecoding for a peptide or polypeptide according to the invention may beused in gene therapy.

The medicament of the invention is usually, but not necessarily (forinstance proteins produced by genetically-modified organisms which areused for food, feed, or to which said subject is exposed to systemic orinhalation route), a (pharmaceutical) formulation comprising as activeingredient at least one of the peptides or polypeptides of theinvention, a gene therapeutic vector capable of expressing said peptideor polypeptide. Apart from the active ingredient(s), such formulationwill comprise at least one of a (pharmaceutically acceptable) diluent.Typically, pharmaceutically acceptable compounds can be found in, e.g.,a Pharmacopeia handbook (e.g. US-, European- or InternationalPharmacopeia). The medicament or pharmaceutical composition of theinvention normally comprises a (prophylactically or therapeutically)effective amount of the active ingredient(s) wherein the effectivenessis relative to the condition or disorder to be prevented or treated.

The medicament or pharmaceutical composition of the invention may needto be administered to a subject in need as part of a prophylactic ortherapeutic regimen comprising multiple administrations of saidmedicament or composition. Said multiple administrations usual occursequentially and the time-interval between two administrations can varyand will be adjusted to the nature of the active ingredient and thenature of the condition to be prevented or treated. The amount of activeingredient given to a subject in need of a single administration canalso vary and will depend on factors such as the physical status of thesubject (as for instance weight and age), the status of the condition tobe prevented or treated, and the experience of the treating doctor,physician or nurse.

The term “diluents” refers for instance to physiological salinesolutions. The term “pharmaceutically acceptable carrier” means anymaterial or substance with which the active ingredient is formulated inorder to facilitate its application or dissemination to the locus to betreated, for instance by dissolving, dispersing or diffusing the saidcomposition, and/or to facilitate its storage, transport or handlingwithout impairing its effectiveness. They include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents (forexample phenol, sorbic acid, chlorobutanol), isotonic agents (such assugars or sodium chloride) and the like. Additional ingredients may beincluded in order to control the duration of action of the activeingredient in the composition. The pharmaceutically acceptable carriermay be a solid or a liquid or a gas which has been compressed to form aliquid, i.e. the compositions of this invention can suitably be used asconcentrates, emulsions, solutions, granulates, dusts, sprays, aerosols,suspensions, ointments, creams, tablets, pellets or powders. Suitablepharmaceutical carriers for use in said pharmaceutical compositions andtheir formulation are well known to those skilled in the art, and thereis no particular restriction to their selection within the presentinvention. They may also include additives such as wetting agents,dispersing agents, stickers, adhesives, emulsifying agents, solvents,coatings, antibacterial and antifungal agents (for example phenol,sorbic acid, chlorobutanol), isotonic agents (such as sugars or sodiumchloride) and the like, provided the same are consistent withpharmaceutical practice, i.e. carriers and additives which do not createpermanent damage to mammals. The pharmaceutical compositions of thepresent invention may be prepared in any known manner, for instance byhomogeneously mixing, coating and/or grinding the active ingredients, ina one-step or multi-steps procedure, with the selected carrier materialand, where appropriate, the other additives such as surface-activeagents. They may also be prepared by micronisation, for instance in viewto obtain them in the form of microspheres usually having a diameter ofabout 1 to 10 μm, namely for the manufacture of microcapsules forcontrolled or sustained release of the active ingredients.

Peptides or polypeptides, homologues or derivatives thereof according tothe invention (and their physiologically acceptable salts orpharmaceutical compositions all included in the term “activeingredients”) may be administered by any route appropriate to thecondition to be prevented or treated and appropriate for the compounds,here the peptide or polypeptide to be administered. Possible routesinclude regional, systemic, oral (solid form or inhalation), rectal,nasal, topical (including ocular, buccal and sublingual), vaginal andparenteral (including subcutaneous, intramuscular, intravenous,intradermal, intraarterial, intrathecal and epidural). The preferredroute of administration may vary with for example the condition of therecipient or with the condition to be prevented or treated.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as solution or a suspension in an aqueous liquid ora non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, electuary or paste. A tablet may be made bycompression or molding, optionally with one or more accessoryingredients. Compressed tablets may be prepared by compressing in asuitable machine the active ingredient in a free-flowing form such as apowder or granules, optionally mixed with a binder, lubricant, inertdiluent, preservative, surface active or dispersing agent. Mouldedtablets may be made by moulding in a suitable machine a mixture of thepowdered compound moistened with an inert liquid diluent. The tabletsmay optionally be coated or scored and may be formulated so as toprovide slow or controlled release of the active ingredient therein.

Viral vectors for the purpose of gene therapy or gene vaccination arehighly amenable to modifications by means of recombinant nucleic acidtechnology. In view of the above, a skilled person will further easilyenvisage that the elimination of the viral vector NKT-cell epitope asapplied in the peptides or polypeptides and their uses according to theinvention can be introduced immediately in the viral vector itself.Hence, the invention further encompasses modified viral vectors definedas isolated viral vectors characterized in that CD1d binding motifs havebeen eliminated by aminoacid substitution or deletion.

The present invention will now be illustrated by means of the followingexamples, which are provided without any limiting intention.Furthermore, all references described herein are explicitly includedherein by reference.

EXAMPLES Example 1: Coagulation Factor VIII

Patients suffering from hemophilia A lack sufficient amounts of factorVIII (FVIII), which is the reason for uncontrolled bleeding tendency.Such patients are treated by infusions of FVIII purified from plasmasource or produced by recombinant technology. Administration of FVIIIresults in the formation of specific antibodies, which in more or less30% of the cases inhibit the function of FVIII as a coagulationcofactor.

Using an algorithm, we identified within the sequence of the FVIIImolecule 3 sequences bearing a CD1d binding sequence, which matched the[FWTHY]-X₂X₃-[ILMV]-X₅X₆-[FWTHY] sequence motif. These motifs arelocated in the A1 and A3 domains, respectively:

FCHISSH in A1 domain (aminoacids 309-315, SEQ ID1)

FWKVQHH in A3 domain (aminoacids 1816-1822, SEQ ID2)

FHAINGY in A3 domain (aminoacids 1918-1924, SEQ ID3)

These sequences have in common (underlined) an aromatic residue(phenylalanine, F) in position 1, an aliphatic residue (isoleucine, I,or valine, V) in position 4, and an aromatic residue (histidine, H, ortyrosine, Y) in position 7.

To determine whether these sequences could activate NKT cells in vivo,FVIII (2 IU) was injected intravenously to hemophilia A mice on 4occasions separated by a 1-week interval. Hemophilia A mice produce noFVIII due to a stop codon introduced in the FVIII gene in exon 16.

Mice were sacrificed 10 days after the last injection, the spleen wasremoved and CD4+ T cells were prepared by magnetic bead sorting. NKTcells are characterized by expression of CD4 and recognition of antigenpresented by CD1d molecule. A tetramer of CD1d was obtained from acommercial supplier and loaded with 15 aminoacid long FVIII peptides,which included peptides containing SEQ ID1, SEQ ID2 and SEQ ID3.Significant binding of CD1d tetramers loaded with these peptides wasobserved, indicating that these 3 peptides were able to bind to CD1d andthat injection of FVIII elicited activation of NKT cells. Representativeresults are given in FIG. 1.

Further, direct immunization with peptides containing a CD1d motif(peptides of SEQ ID1, SEQ ID2 or SEQ ID3) was sufficient as to elicitthe activation of NKT cells and production of antibodies to FVIII.Prominent activation was observed with peptide of SEQ ID1.Representative results are given in FIG. 2.

Bone marrow chimeras were constructed in which hemophilia A mice werefirst irradiated and reconstituted with the bone marrow of mice lackingNKT cells, namely CD1d knocked-out mice. In the absence of NKT cells,mice were unable to produce significant amounts of antibodies to FVIII,and virtually no antibodies inhibiting the function of FVIII (FIG. 3).

The A1 domain of FVIII was produced by recombinant technology in itsnatural sequence or with a substitution of F309 and H315 by serine(polypeptide of SEQ ID4). FVIII A1 domains in natural sequence or SEQID4 were used to immunize separate groups of mice. The results showedthat substitution of F309 and H315 by S (SEQ ID4) was sufficient toprevent activation of NKT cells as assessed from spleen CD4+ T cells asdescribed above.

The A3 domain of FVIII was produced by recombinant technology in itsnatural sequence or with a substitution of F1816 and H1822 by serine(polypeptide of SEQ ID 5). FVIII A3 domains in natural sequence or SEQID5 were used to immunize separate groups of mice. The results showedthat substitution of F1816 and H1822 by S (SEQ ID5) was sufficient toprevent activation of NKT cells as assessed from spleen CD4+ T cells asdescribed above.

A B domain-deleted FVIII molecule in its natural sequence elicitedactivation of NKT cells (see above). A FVIII molecule with 4 aminoacidsubstitutions was prepared containing F309S, H315S, F1816S and F1918S(SEQ ID6). Intravenous injections of such mutated FVIII in hemophilia Amice did not result in the formation of antibodies to FVIII and,consequently, no antibodies inhibiting the function of FVIII.

It was therefore concluded that:

(1) FVIII naturally contains several CD1d binding motifs;

(2) these CD1d motifs are functional and elicit activation of NKT cells;

(3) activation of NKT cells is a requisite for the production ofantibodies to FVIII;

(4) elimination of the CD1d motifs by aminoacid substitution issufficient to eliminate the production of anti-F VIII antibodies

It should be clear for those skilled in the art that the invention alsoapplies to animals made transgenic for the production of coagulationfactors such as factor IX.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1

NKT cells recognize factor VIII epitopes presented by CD1d

Hemophilia A mice were immunized with 2 IU Factor VIII on 4 occasionsseparated by one week. The spleen was then removed and CD4+ T cells wereprepared by magnetic bead sorting. A fluorochrome-labeled tetramer ofCD1d was obtained from a commercial supplier and loaded with 15aminoacid long FVIII peptides, which included peptides containing SEQID1, ID2 and ID3. Loading was carried out at room temperature overnightin the dark.

Tetramers were then incubated for 30 minutes at 4° C. with the CD4+ Tcell population and the cell suspension was analyzed by Facs.

The figure shows that CD1d tetramers loaded with peptide of SEQ ID1(44pept in the figure) are recognized by NKT cells. CD1d ctl(−) showsthe % of NKT cells recognizing unloaded tetramers. CD1d ctl (+) showsthe % of NKT cells recognizing tetramers loaded with alpha-gal ceramide,which recruits all NKT cells. Up to 45% of NKT cells recognize 44pept,which is compatible with the absence of polymorphism at CD1d level andvery limited polymorphism at the level of the NKT T cell receptor.

FIG. 2

Immunization of hemophilia A mice with a CD1d-restricted peptideselicits anti-factor VIII antibodies

Hemophilia A mice were immunized 3 times subcutaneously with 50 μg of anequimolar mixture of peptides of SEQ IDI (44pept) and peptide of SEQ ID2(256pept) adsorbed on aluminum hydroxyde. A control group receivedphysiological serum instead of peptides. Plasma was taken 10 days afterthe last immunization and assessed for the presence of anti-Factor VIIIantibodies, using a direct binding assay. Briefly, Factor VIII (10IU/ml) was insolubilized on polystyrene plates, which were washed andincubated with a 1/10 dilution of plasma. After a further washing, aHRP-labeled goat anti-mouse antiserum was added, followed by an enzymesubstrate. Colour development was read as OD.

The figure shows that hemophilia A mice immunized with peptides of SEQID1 and of SEQ ID2 develop antibodies to Factor VIII.

FIG. 3

Hemophilia A mice reconstituted with bone marrow from CD1d KO mice donot produce antibodies to Factor VIII

Hemophilia A mice were lethally irradiated and reconstituted with thebone marrow of CD1d KO mice (5×10⁶/mouse), which lack NKT cells. Sixweeks after bone marrow reconstitution the mice received 4 IV injectionsof 2 IU/ml separated by one week. Mice were bled 10 days after the lastimmunization and the plasma was assayed for the presence of anti-FactorVIII antibodies using a direct binding assay as described in the legendof FIG. 2. A control group of irradiated hemophilia A mice wasreconstituted with a normal bone marrow.

The figure indicates that, although control mice produce highconcentrations of anti-FVIII antibodies after the fourth injection ofFactor VIII (left panel), mice reconstituted with the bone marrow of NKTcell deficient mice did not (right panel).

Example 2: Adenovirus 5 Viral Vectors

Viral vectors are commonly used for gene therapy and gene vaccination.One of the most common of these viral vectors is derived fromadenovirus, serotype 5. Adenoviruses (Ad) are non-enveloped virusespossessing a linear, double-stranded DNA genome of about 35 kb. HumanAd5 has a capsid consisting of 3 major structural proteins: hexon,penton, and fiber. Neutralizing antibodies are raised towards hexonproteins. Such antibodies are very common in humans as a consequence ofviral infection. The presence of such antibodies blocks the entry of theviral vector and, consequently, prevents expression of the transgeneprotein carried by the vector. Anti-Ad5 antibodies are generated in thecourse of an adaptive response, which depends on activation of CD4+ Tcells specific for epitopes presented in the context of MHC class IImolecules.

It is known that Ad5 activates the innate immune system, though theprecise mechanism by which it occurs and the location where it takesplace remain unclear. Yet, activation of the innate immune system couldbe a required step for neutralizing antibodies to be formed.

Using algorithms, we identified 7 aminoacid sequences matching with thegeneral motif [FWTHY]-X₂X₃-[ILMV]-X₅X₆-[FWTHY] of a CD1d bindingsequence (SEQ ID7, with motifs underlined) in hexon 6.

Mice were injected intravenously with 10⁹ PFU Ad5 vector on 3 occasionsat 10-day intervals. CD4+ T cells were then prepared from the spleen bymagnetic bead sorting. CD4+ T cells were incubated with CD1d tetramersloaded with peptides corresponding to each of the 7 sequencesidentified. It showed that a significant proportion (±10%) of CD4+ NKTcells were labeled by tetramers, indicating that Ad5 vector injectionsactivated NKT cells specific for the peptide of SEQ ID7. In addition,such mice produced specific antibodies of the IgG2a isotype,characteristic of neutralizing antibodies in the mouse.

A viral vector was prepared which contained a substitution of [FW] byserine S for each of the 7 aminoacid sequences identified. This mutatedviral vector (SEQ ID8, with underlined motifs) was used to immunizeanimals according to the same protocol as described above for thenatural sequence. The proportion of NKT cells as assessed usingtetramers loaded with the peptide in natural sequence (SEQ ID7) was <1%and the concentration of Ad5 virus specific antibodies was significantlyreduced (up to 10-fold).

It was therefore concluded that substitution of F to S in each P1location of CD1d binding motifs was sufficient as to reduce NKT cellactivation and thereby reduce the production of anti-Ad5 antibodies.

Example 3: Genetically-Modified Proteins

Proteins to which subjects are exposed by way of inhalation or ingestionare frequently eliciting unwanted reactions in predisposed subjects.Allergic asthma affects millions of people across the world. Foodallergy on the other hand has an overall prevalence of ±2.5% in thegeneral population. Allergens either airborne, ingested or penetratingthe skin could share properties by which they activate NKT cells.

One of the most common food allergen is apple (Malus domesticus), andallergenicity is almost exclusively borne by the Mal d 1 protein, a 159aminoacid long protein, which protects the plant against infectiousagents. A sequence motif was identified using computer algorithms, whichcorresponds to the general motif [FWTHY]-X₂X₃-[ILMV]-X₅X₆-[FWTHY] of aCD1d binding sequence.

FKLIESY corresponding to aminoacids 144-150 of Mal d 1 (SEQ ID9)

A recombinant form of Mal d 1, in which F144 and Y150 were mutated in Swas produced by genetic engineering. The recombinant form of Mal d 1therefore encompasses peptide of sequence:

SKLIESS (SEQ ID10)

Synthetic peptides corresponding to SEQ ID9 and SEQ ID10 were produced.Their capacity to activate NKT cells was determined in vitro using humandendritic cells derived from peripheral blood monocytes of an individualsensitized to Mal d 1. Dendritic cells loaded with each one of the twopeptides were incubated in the presence of NKT cells obtained from thesame individual by sorting peripheral lymphocytes using specific markerssuch as CD4 and NKG2D. It was observed that NKT cells incubated withpeptide of SEQ ID9 activated a significant proportion of NKT cells,while the mutated peptide of SEQ ID10 did not. Additionally, human CD1dtetramers loaded with peptides of SEQ ID9 were recognized by asignificant proportion of NKT cells, but tetramers loaded with themutated peptide of SEQ ID10 were recognized by less than 1% of NKTcells.

The two F144S and Y150S mutations are introduced directly in clonalcells by site-directed mutagenesis. The full organism is then producedby conventional growth strategies. Apples produced by this GMO do notelicit allergic reactions.

One specific application of the peptides or polypeptides of the presentinvention is celiac disease (gluten intolerance). This disease is amongthe most commons in human beings and is related to T cell activation togliadin epitopes which are presented in the context of MHC class IIdeterminants. A genetic susceptibility has been described, with humanbeings carrying the HLA-DQ2 or DQ8 class II determinant beingpredisposed to disease. These class II determinants present peptideswhich have been submitted to deamidation by transglutaminase. However,these events are the results of intestinal inflammatory reaction, likelyrelated to the innate immune system.

Gliadins are monomers of 250-300 aminoacid residues. A search for thegeneral motif [FW]-XX-[ILM]-XX-[FWTHY] of a CD1d binding sequence usingcomputer algorithms identified such sequence (SEQ ID11, see listing ofsequences) in alpha-gliadin. A mutated form of alpha-gliadin was thenproduced in which the F residue of the motif was substituted by a Sresidue (SEQ ID12, see addendum).

The same procedure as for Mal d 1 was followed to show that, althoughpolypeptide of SEQ ID11 activated a significant proportion of NKT cellswhen presented by antigen-presenting dendritic cells, the mutated formof the polypeptide (SEQ ID12) failed to do so. As for Mal d 1, humanCD1d tetramers loaded with a synthetic peptide representing the motifidentified in the polypeptide of

SEQ ID11 were recognized by NKT cells, while tetramers loaded with themutated form of the motif as shown in SEQ ID12 were not.

The mutation was introduced directly in clonal cells by site-directedmutagenesis. The full organism was then produced by conventional growthstrategies. Cereals containing the mutated form of gliadin do not elicitreactions of intolerance.

It should be obvious for those skilled in the art that the presentinvention can also be applied to proteins which are added to, forinstance, genetically-modified organisms to increase their resistance toinsecticides, pesticides or any other modifications judged to bebeneficial. Such modifications carry the risk of creating new CD1dbinding motifs.

Additional examples of genetically-modified proteins with reducedallergenicity/immunogenicity are:

-   -   food allergens such as soybean, peanut and fruits of the        Rosaceous family    -   milk proteins    -   airborne allergens such as latex (Hevea brasiliensis), pollens        of grasses such as Rye grass (Lolium perenne), Timothy (Phleum        pratense) or Kentucky blue grass (Poa pratensis)    -   fish parvalbumin    -   honey bee phospholipase A2

It should also be clear for the one skilled in the art that theinvention extends to methods by which peptides or polypeptides of theinvention are produced, including the production of transgenic plantsand animals.

Example 4: Allergen Der p 1

Der p 1 is a cysteine protease which is the main allergen of theso-called house dust mite (HDM), D. pteronyssinus. Sensitization to HDMis by far the commonest trigger of allergic asthma and rhinitisworldwide. Der p 1 contains 3 motifs matching the general CD1d bindingmotif [FWTHY]-X₂X₃-[ILMV]-X₅X₆-[FWTHY], as identified using computeralgorithms and which are:

SEQ ID13: FSGVAAT aminoacids 38-44 of Der p 1

SEQ ID14: HSAIAAVI aminoacids 135-141 of Der p 1

SEQ ID15: YPYVVIL aminoacids 216-222 of Der p 1

Peptides of SEQ ID13, SEQ ID14 and SEQ ID15 were synthesized and used toload CD1d tetramers.

BALB/c mice were submitted to intranasal administration of Der p 1,using 50 μl of saline containing 100 μg of Der p 1. This challengeprocedure was repeated twice on three consecutive days at one-weekinterval. The mice were sacrificed 5 days after the last nasalinstillation and the spleen was removed. CD4+ T cells were purified bymagnetic bead sorting and incubated in the presence of the CD1dtetramers loaded with peptides of SEQ ID13, SEQ ID14 or SEQ ID15. Byfluorescence-activated cell sorter (facs) determination, it was observedthat a significant percentage of cells (±10%) were stained with thetetramers, identifying them as CD4+ NKT cells. It was thereforeconcluded that peptides of SEQ ID13, SEQ ID14 and SEQ ID15 werefunctional in binding to CD1d and in being recognized by NKT cells.

CD4+ T cells obtained from the above experiments were incubated inculture medium in the presence of an antigen-presenting cell whichexpresses the CD1d molecule. Such cells are commercially available, asfor instance the JAWS2 cells, which do not express MHC class IIdeterminants. JAWS2 cells were loaded with Der p 1 and presentation ofDer p 1-derived epitopes by CD1d was evaluated by measuring theproduction of cytokines such as IFN-gamma and IL-4 as markers of NKTactivation. It could be observed that a significant production ofcytokines was present, confirming that Der p 1 contained epitopespresented by CD1d molecules.

Next, a mutated form of Der p 1 was prepared by genetic engineering, inwhich the 3 aminoacid residues predicted to be in position P1 for CDbinding of peptides of SEQ ID13, SEQ ID14 and SEQ ID15 were substitutedby serine. The mutated Der p 1 (SEQ ID16) was used for nasalinstillation as described above with Der p 1 in natural sequence (SEQID17). In such a case, no significant binding of CD4+ T cell splenocyteswas observed when incubated with the tetramers loaded with peptide ofSEQ ID13, peptide of SEQ ID14 or peptide of SEQ ID15, indicating thatthe mutated Der p 1 had lost its capacity to activate NKT cells specificfor these peptides.

Further, mutated Der p 1 (SEQ ID16) was used to load JAWS2 cells andtested for its capacity to activate NKT cells. For this experiment, NKTcells were used as obtained from mice immunized with Der p 1 in eithernatural or mutated configuration. The production of IFN-gamma and IL-4was taken as an indication of NKT activation. It was observed that NKTcells obtained from mice immunized with natural sequence Der p 1 failedto be activated when incubated in the presence of JAWS2 cells loadedwith mutated Der p 1.

It was therefore concluded that Der p 1 in natural sequence containedfunctional CD1d restricted T cell epitopes activating NKT cells.Further, elimination of such functional CD1d-restricted epitopes bymutation was sufficient to eliminate NKT cell activation.

Example 5: Antibodies

Antibodies are used as therapeutic agents in a large number ofindications, from chronic inflammatory diseases such as rheumatoidarthritis (e.g., anti-TNF-alpha antibodies) or allergic asthma (e.g.anti-IgE antibodies), to tumors (e.g., anti-CD20 antibodies). More than120 therapeutic antibodies are presently used for clinical applicationsat various stages from preclinical to phase III trials and accepted forroutine clinical practice.

Therapeutic antibodies are either chimeric or fully humanized, whichcontains sequence of foreign origin only in the complementaritydetermining regions of the variable parts. A minority of such antibodiesare derived from the human repertoire and, as such, considered as poorlyimmunogenic. However, antibodies towards the therapeutic antibody, evenwhen directly derived from the human repertoire, are produced by amajority of the patients under treatment, with, in a significantproportion of the cases, the production of antibodies neutralizing theactivity of the therapeutic agent.

A search for epitopes matching the CD1d binding motif in human IgGantibody sequence was carried out using computer algorithms. One of suchmotif was identified in the CH2 region (second domain of the heavy chainconstant part) of each of the 4 IgG subclass (IgG1, IgG2, IgG3 and IgG4)and a second motif was identified in the CH3 loop of IgG1, IgG2 andIgG4:

SEQ ID 18: YRVVSVL (CH2 of IgG1 and IgG4) SEQ ID 19:FRVVSVL (CH2 of IgG2 and IgG3) SEQ ID 20:HEALHNH (CH3 loop of IgGL IgG2 and IgG4)

Synthetic peptides corresponding to SEQ ID18, SEQ ID19 and SEQ ID20 wereproduced and used to load human CD1d tetramers as for the examples above(see for instance example 4 for allergen Der p 1). Peripheral bloodcells were obtained by venous puncture of patients who had received aninjection of a therapeutic antibody during the previous 5 days. CD4+ Tcells were purified by magnetic bead sorting. The cells were thenincubated with tetramers loaded with peptides of SEQ ID18, SEQ ID19 orSEQ ID20. Analysis by facs identifies a significant proportion of NKTcells (±10%) labeled by tetramers.

Monoclonal human antibodies of the IgG4 isotype were derived from theperipheral blood B lymphocytes by transformation with the Epstein-Barrvirus. The genomic sequence of such antibodies was obtained fromtransformed B cells. A viral vector containing the corresponding cDNAsequence was constructed and used for transfection of CHO cells. Allthese methods are known in the art (see for instance, Jacquemin et alBlood 92: 496-506, 1998).

The hydrophobic aminoacid residues located in position 1 in the peptidesof SEQ ID18 and SEQ ID20 were mutated to a serine and the mutatedantibody produced by transfected CHO cells.

Peripheral blood CD4+ T cells obtained as above were exposed in culturemedium to human dendritic cells (derived from human peripheral bloodmonocytes by methods known in the art) and loaded with either theantibody in natural configuration (SEQ ID21) or its mutated counterpart(SEQ ID22). After culturing the cells with CD4+ T cells for 5 to 7 days,the population of CD4+ T cells activated by either natural or mutatedantibody was evaluated. CD4+ NKT cells were separated from CD4+ T cellsusing an antibody to NKG2D, a surface marker associated with NK or NKTcells only.

It was observed that CD4+ T cells and NKT cells were activated when theantibody in natural sequence was used (SEQ ID21), while the mutated formof the antibody (SEQ ID22) only activate class II restricted CD4+ Tcells and not NKT cells.

It was concluded that human IgG antibodies contained epitopescorresponding to the [FWTHY]-X₂X₃-[ILMV]-X₅X₆-[FWTHY] motif, having thecapacity to be recognized by and to activate NKT cells. Further,mutation of key hydrophobic aminoacid residues within such motif wassufficient to prevent activation of NKT cells.

It should be understood that the examples provided here are notexhaustive and that combinations of proteins or peptides containingvarious numbers of aminoacid substitutions or deletions can beenvisioned. For instance, in example 1, various combinations ofsubstitution of hydrophobic aminoacids can be delineated.

SEQ ID 1  Factor VIII aminoacids 309-315 (human)  FCHISSH  SEQ ID 2 Factor VIII aminoacids 1816-1822 (human)  FWKVQHH  SEQ ID 3 Factor VIII aminoacids 1918-1924 (human)  FHAINGY  SEQ ID 4 Factor A1 domain (mutations F309S and H315S underlined) (human)    1ATRRYY LGAVELSWDYMQSDLGELP VDAR FPP RV P K S FPF   41NTS VVYKKT LFVE F T VHLFNI AKPR P PWMGLLGPTI QA EV   81YDT VVITLKNMASHPVSLHAVGVS Y W K ASEGAEYDDQTSQ  121REK EDDK VFPGGSHTYVWQVLKE N G P MASDPLCLTYSYLS  161HVD LVK DLNSGLIG AL LVCRE GSLA K E KTQ TL HKFILLFA  201VFD EGKSWHSE TKN SLMQDRDAASARAWPKWITVNGYVNR  241SLP GLIGCH R KSV YWH VIG MGTT PEV HSIF LEG HTFL VRN  281HRQ AS LEI SPIT FLT AQTLLM DL GQFL LSC HIS SS QH DGM  321EAY VKV DS CPEEP QLRMKNNE EAED YDDDLTDSEMDVVRF  361D DDN SPSFI Q IRS VA KK HPKTW VHYIA AEEEDW DYAP LVL  401APDDR SYKS QYLN NGPQ RIGRK YKK VRF MAYT DETFKTRE  441AIQ H ES G ILGPLLYGE VG D TLL II FK NQ AS RP YNI YP H GI  481TD VR PLYSR R LPK GVKHLK DFP ILP GEIFK YK WTV TVEDG  521PTK SDPRCLTRYYS SFVNMERDLASG  SEQ ID 5 Factor VIII A3 domain (mutations F1816S and Hi822S underlined) (human) 1637 SQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKED  1677FDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPH  1717VLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHL  1757GLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQR  1798QGAEPRKNFVKPNETKTYSWKVQHSMAPTKDEFDCKAW  1836AYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQE  1876FALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKEN  1916YRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIFIS  1956IHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGI  1996WRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGEIMDF  2036QITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDL  2076LAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTY  2116RGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLIIPTH  2156YSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSY  2196FTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQ  2236KTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFF  2276QNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVH  2316 QIALRMEVLGCEAQDLY* SEQ ID 6 Factor VIII (mutations F309S, H315S, F1816S and F1918S underlined) (human) ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLSCHISSSQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQUESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYEGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYSWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRSHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY*GWPLQHLPLPSPLPPQLQGSVPPWLAFYLCAKS*QTLP*SLL SEQ ID 7 Hexon, Human adenovirus 5, (CD1d binding motifs underlined): (virus) MATPSMMPQWSYMHISGQDASEYLSPGLVQFARATETYFSLNNKFRNPTVAPTEEDVTTDRSQRLTLRFIPVDREDTAYSYKARFTLAVGDNRVLDMASTSFDIRGVLDRGPTFKPYSGTAYNALAPKGAPNPCEWDEAATALEINLEEEDDDNEDEVDEQAEQQKTHVFGQAPYSGINITKEGIQIGVEGQTPKYADKTFQPEPQIGESQWYETEINHAAGRVLKKTTPMKPCYGSYAKPTNENGGQGILVKQQNGKLESQVEMQFFSTTEAAAGNGDNLTPKVVLYSEDVDIETPDTHISYMPTIKEGNSRELMGQQSMPNRPNYIAFRDNFIGLMYYNSTGNMGVLAGQASQLNAVVDLQDRNTELSYQLLLDSIGDRTRYFSMWKQAVDSYDPDVRIIENHGTEDELPNYCFPLGGVINTETLTKVKPKTGQENGWEKDATEFSDKNEIRVGNNFAMEINLNANLWRNFLYSNIALYLPDKLKYSPSNVKISDNPNTYDYMNKRVVAPGLVDCYINLGARWSLDYMDNVNPFNEEHRNAGLRYRSMLLGNGRYVPFHIQVPQKFFAIKNLLLLPGSYTYEWNFRKDVNMVLQSSLGNDLRVDGASIKFDSICLYATFFPMAHNTASTLEAMLRNDTNDQSFNDYLSAANMLYPIPANATNVPISIPSRNWAAFRGWAFTRLKTKETPSLGSGYDPYYTYSGSIPYLDGTFYLNHTFKKVAITFDSSVSWPGNDRLLTPNEFEIKRSVDGEGYNVAQCNNITKDWFLVQMLANYNIGYQGFYIPESYKDRMYSFFRNFQPMSRQVVDDTKYKDYQQVGILHQHNNSGFVGYLAPTMREGQAYPANFPYPLIGKTAVDSITQKKFLCDRTLWRIPFSSNFMSMGALTDLGQNLLYANSAHALDMTFEVDPMDEPTLLYVLFEVFD VVRVHRPHRGVIETVYLRTPFSAGNATT SEQ ID 8 Hexon, Human adenovirus 5 (mutations of P1 anchoring residue underlined). (virus) MATPSMMPQWSYMHISGQDASEYLSPGLVQFARATETSFSLNNKFRNPTVAPTHDVTTDRSQRLTLRFIPVDREDTAYSYKARFTLAVGDNRVLDMASTSFDIRGVLDRGPTFKPYSGTASNALAPKGAPNPCEWDEAATALEINLEEEDDDNEDEVDEQAEQQKTHVFGQAPSSGINITKEGIQIGVEGQTPKYADKTFQPEPQIGESQWYETEINHAAGRVLKKTTPMKPCYGSYAKPTNENGGQGILVKQQNGKLESQVEMQFFSTTEAAAGNGDNLTPKVVLYSEDVDIETPDTHISYMPTIKEGNSRELMGQQSMPNRPNYIAFRDNSIGLMYYNSTGNMGVLAGQASQLNAVVDLQDRNTELSYQLLLDSIGDRTRYFSMWKQAVDSYDPDVRIIENHGTEDELPNYCFPLGGVINTETLTKVKPKTGQENGWEKDATEFSDKNEIRVGNNFAMEINLNANLWRNFLSSNIALYLPDKLKYSPSNVKISDNPNTYDYNINKRVVAPGLVDCYINLGARWSLDYMDNVNPFNHHRNAGLRSRSMLLGNGRYVPFSIQVPQKSFAIKNLLLLPGSYTYEWNFRKDVNMVLQSSLGNDLRVDGASIKSDSICLYATFFPMAHNTASTLEANILRNDTNDQSFNDYLSAANNILYPIPANATNVPISIPSRNWAAFRGWASTRLKTKETPSLGSGYDPYYTYSGSIPYLDGTFYLNHTSKKVAITFDSSVSWPGNDRLLTPNEFEIKRSVDGEGYNVAQCNNITKDSFLVQMLANYNIGYQGFYIPESYKDRMYSFFRNFQPMSRQVVDDTKYKDSQQVGILHQHNNSGFVGYLAPTMREGQAYPANFPSPLIGKTAVDSITQKKFLCDRTLWRIPFSSNSMSMGALTDLGQNLLYANSAHALDMTFEVDPMDEPTLLYVLFEVSD VVRVHRPSRGVIETVYLRTPFSAGNATT SEQ ID 9 Mal d 1, malus domesticus, aminoacids 144-150  FKLIESY  SEQ ID 10 Mal d 1, malus domesticus, F144S and Y150S mutations underlined (vegetal) SKLIESS  SEQ ID 11  Alpha-Gliadin (CD1d binding motif underlined) MVRVPVPQLQPQNPSQQQPQEQVPLVQQQQFPGQQQPFPPQQPYPQPQPFPSQQPYLQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPFRPQQPYPQSQPQYSQPQQPISQQQQQQQQQQQQKQQQQQQQQILQQILQQQLIPCRDVVLQQHSIAYGSSQVLQQSTYQLVQQLCCQQLWQIPEQSRCQAIIINVVHAIILHQQQQQQQQQQQQPLSQVSFQQPQQQYPSGQGSFQPSQQNPQAQGSVQPQQLPQFEEIRNLALETLPAMCNVYIPPYCTIAPVGIFGT NYRSEQ ID 12  Alpha-Gliadin (mutation underlined) MVRVPVPQLQPQNPSQQQPQEQVPLVQQQQFPGQQQPFPPQQPYPQPQPFPSQQPSLQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPFRPQQPYPQSQPQYSQPQQPISQQQQQQQQQQQQKQQQQQQQQILQQILQQQLIPCRDVVLQQHSIAYGSSQVLQQSTSQLVQQLCCQQLWQIPEQSRCQAISNVVHAIILHQQQQQQQQQQQQPLSQVSFQQPQQQYPSGQGSFQPSQQNPQAQGSVQPQQLPQSEEIRNLALETLPAMCNVYIPPSCTIAPVGIFGT NYRSEQ ID 13 D. pteronyssinus Der p 1, aminoacids 38-44 (pyroglyphidae, Dermatophagoides pteronyssinus, European house dust mite)  FSGVAAT  SEQ ID 14 D. pteronyssinus Der p 1, aminoacids 135-141 (pyroglyphidae, Dermatophagoides pteronyssinus, European house dust mite)  HSAIAAVI  SEQ ID 15 D. pteronyssinus Der p 1, aminoacids 216-222 (pyroglyphidae, Dermatophagoides pteronyssinus, European house dust mite)  YPYVVIL  SEQ ID 16 Mature Der p 1 (mutations of P1 anchoring residues F38S, H135S and Y216S underlined) (pyroglyphidae, Dermatophagoides pteronyssinus, European house dust mite) ETNACSINGNAPAEIDLRQMRTVTPIRMQGGCGSCWASSGVAATESAYLAYRNQSLD LAEQELVDCASQHGCHGDTI PRGIEYIQHNGVVQESYYRYVAREQSCRRPNAQRFGISNYCQTYPPNVNKIREALAQT SSAIAVIIGIKDLDAFRHY DGRTIIQRDNGYQPNYHAVNIVGYSNAQGVDYWIVRNSWDTNWGDNGYGYFAANI  DLMMIEESPYVVIL SEQ ID 17 Mature Der p 1 (CD1d epitopes underlined): (pyroglyphidae, Dermatophagoides pteronyssinus, European house dust mite) ETNACSINGNAPAEIDLRQMRTVTPIRMQGGCGSCWAFSGVAATESAYLAYRNQSLD LAEQELVDCASQHGCHGDTI PRGIEYIQHNGVVQESYYRYVAREQSCRRPNAQREGISNYCQTYPPNVNKIREALAQT HSAIAVIIGIKDLDAFRHY DGRTIIQRDNGYQPNYHAVNIVGYSNAQGVDYWIVRNSWDTNWGDNGYGYFAANI  DLMMIEEYPYVVIL SEQ ID 18  IgG antibody, CH2 domain of IgG1 and IgG4 (human)  YRVVSVL SEQ ID 19  IgG antibody, CH2 domain of IgG2 and IgG3 (human)  FRVVSVL SEQ ID 20  IgG antibody, CH3 domain of IgG1, IgG2 and IgG4 (human) HEALHNH  SEQ ID 21 Human IgG4 FC fragment (CD1d epitopes underlined) (human) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNANYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFS CSVNIFHALHNHYTQKSLSLSLGK SEQ ID 22 Human IgG4 FC fragment (mutated aminoacids underlined) (human) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTSRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFS CSVMSEALHNHYTQKSLSLSLGK

1-14. (canceled)
 15. An engineered peptide derived from a peptideselected from the group consisting of an allofactor, a viral vector, agenetically-modified organism and a vaccine for an allergen, the peptidenaturally comprising one or more motif consisting of[FWTHY]-X₂X₃-[ILMV]-X₅X₆-[FWTHY] and the peptide being capable ofbinding to CD1d molecule and/or activating NKT cells, the engineeredpeptide having been modified to eliminate the [FWTHY] amino acids atpositions P1 and P7 of the motif comprised in the peptide bysubstitution of the [FWTHY] amino acid by an amino acid different from[FWTHY], so that the binding to CD1d molecule is reduced in theengineered peptide as compared with the peptide.
 16. The engineeredpeptide of claim 1 further having a reduced capacity to activate NKTcells as compared with the peptide naturally comprising one or moremotif consisting of [FWTHY]-X₂X₃-[ILMV]-X₅X₆-[FWTHY].
 17. The engineeredpeptide of claim 1 further comprising a substitution of the amino acid[ILMV] at position P4.
 18. The engineered peptide of claim 1, whereinthe allofactor is selected from the group consisting of factor VIII,factor IX, factor X, fibrinolytic factors, enzymes used for thetreatment of Pompe disease, insulin, growth hormone, cytokines andtherapeutic antibodies.
 19. The engineered peptide of claim 1, whereinthe allofactor is Factor VIII, and wherein the modified peptidecomprises SEQ ID NO:4 or SEQ ID NO:5.
 20. A method for the production ofthe engineered peptide of claim 1, comprising the steps of identifyingin a peptide selected from the group consisting of an allofactor, aviral vector, a genetically-modified organism and a vaccine for anallergen one or more motif consisting of[FWTHY]-X₂X₃-[ILMV]-X₅X₆-[FWTHY]; measuring a binding of the peptide toCD molecule and/or a capacity to activate NKT cells, and substitutingthe [FWTHY] amino acids at positions P1 and P7 of the motif by an aminoacid different from [FWTHY], so that, as compared with the peptide, thebinding to CD1d by the engineered peptide is reduced.
 21. The method ofclaim 6 further comprising the step of measuring a reduced capacity toactivate NKT cells of the engineered peptide, as compared with thepeptide naturally comprising one or more motif consisting of[FWTHY]-X₂X₃-[ILMV]-X₅X₆-[FWTHY].